RU2354645C1 - Kreatine amides, method for its making, neuroprotective agent - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention refers to pharmaceutical chemistry, namely to new biologically active substances (BAS) and to their properties, namely to kreatine derivatives of general formula: NH=C(NH₂)-N(CH₃)-CH₂-CO-NH-R∗X, where R is aminoacid residual of aliphatic, aromatic or heteroaromatic amino acid or its derivative, representing pharmaceutically acceptable salts of amino acids, esters of amino acids, amides of amino acids or peptides; X is low-molecular organic or mineral acid, or water. New substances are produced by interaction of guanydilic agents and sarcosine amides in polar organic solvents at temperature 50°C and less.

EFFECT: new compounds can be used as a neuroprotective agent.

3 cl, 10 ex, 6 tbl

Description

The invention relates to the field of pharmaceutical chemistry, namely to new biologically active substances (BAS) and their properties. In particular, the invention relates to creatine derivatives - substances of the general formula

NH = C (NH ₂ ) —N (CH ₃ ) —CH ₂ —CO — NH — R * X, where R is an amino acid residue or a substituted amino acid residue; X is a low molecular weight organic or mineral acid or water.

Creatine (Cr) is an endogenous nutrient present in various tissues of mammals, for example, in the liver, kidneys, muscle tissue, brain tissue, blood, and is both in a free state and in the form of creatine phosphate. Creatine is considered as a means of improving energy tissue metabolism - increasing the energy reserve of ATP, primarily in muscle and nerve cells.

In the mitochondria of the cell, under the action of the creatine kinase enzyme, creatine reversibly interacts with adenosine triphosphate (ATP) to form creatine phosphate and adenosine diphosphate (ADP). This interaction performs the function of maintaining the concentration of ATP at a constant level at the moments of its intense consumption. Other ways - glycolysis or oxidative phosphorylation - replenish ATP reserves much more slowly. When ATP is consumed, a large amount of ADP is released in the cell, which leads to the transfer of orthophosphate from creatine phosphate to ADP and restoration of the initial ratio between ATP and ADP. Due to the high affinity of creatine kinase for ADP, this process proceeds up to a decrease in the concentration of creatine phosphate below several tens of microns.

Creatine phosphate (CrF) during the maintenance of the membrane potential, activation of metabolites or contractile activity of the cell is a reserve of macroergic phosphate. It maintains the level of ATP with increasing energy expenditures in the cell, i.e. returns the orthophosphate residue to ADP. Along with glycogen, KrF is one of the main sources of the high-energy phosphate conversion cycle and, thus, is involved in the oxidative phosphorylation of glucose, which provides the release of energy necessary for the functioning of muscle tissue cells, including skeletal muscle and heart muscle. Due to the fact that creatine phosphate is able to regenerate ATP at a faster rate than is achieved using glycogen, an increase in the amount of creatine in the muscles increases muscle reserves of creatine phosphate, improves the working capacity (endurance) of muscles and increases muscle mass.

Creatine phosphate and creatine are also allosteric regulators of cellular processes. Oral administration of creatine has been shown to increase the total creatine content in the body. So, taking from 20 to 30 g of creatine monohydrate per day for several days leads to an increase of more than 20% in the total creatine content in human skeletal muscle. These properties attract particular attention in connection with the possibility of their use as a food supplement to strengthen the body and increase performance, especially when used as an additive to the diet of athletes. Thus, the use of creatine monohydrate in a daily dose of 15 g for at least 2 days is used to increase muscle strength (WO 94/02127, 1994). Currently, creatine is recommended as a dietary supplement, which is especially important for older people and vegetarians, as these groups have a pronounced tendency to decrease the creatine content in the muscles. Additives are used in the form of a dry powder, liquid, or semi-liquid form (WO 97/45026, 1997). The compositions obtained on their basis are stable in the refrigerator at a temperature of 4 ° C for a long time, but at room temperature they degrade for a week.

Along with the use in the food industry, creatine and creatine phosphate are quite widely used in medicine. So, creatine, kratin phosphate and cyclocreatine (US 6706764, 2004) are recommended for the treatment of diseases of the nervous system, such as diabetic and toxic neuropties, Alzheimer's disease, Parkinson's disease, stroke, etc., metabolic disorders such as hyperglycemia and diabetes mellitus ( US 6193973, 2001). Oral administration of creatine has been described for the treatment of heart and respiratory failure (WO / EP 97/06225, 1999), asthma (US 6093746, 2000). The use of creatine phosphate for the treatment of cardiovascular diseases has been shown, it is promising for the treatment of neoplasms (US 5219846, 1993). However, the use of creatine and creatine phosphate is limited by poor solubility and instability in aqueous media at physiological pH values (RU 2295261, 2007).

Moreover, creatine is poorly absorbed from the gastrointestinal tract - the degree of absorption is 1-14%. This necessitates the use of high doses of creatine. In order for creatine use to be effective, the compositions currently being produced require up to 20 g per day. At the same time, along with an increase in the cost of therapy, the introduction of high doses of creatine can lead to negative consequences for the body - impaired nitrogen metabolism, gastrointestinal upsets, diarrhea, etc.

In this regard, it is of great interest to obtain creatine derivatives with greater stability or higher biological activity, which will allow, on the one hand, to reduce the dose of the substance used, and, on the other hand, to discover new applications.

Derivatives of creatine and various organic acids aroused the greatest interest. So, it is known to use creatine pyruvates (US 6166249, 2000; RU 2114823, 1998) to increase performance, reduce body weight, adapt to conditions of oxygen deficiency in ischemia, as a dietary supplement, to protect the skin from aging and exposure to sunlight (US 7186754 , 2007) in the treatment of female genital disorders, in particular dysmenorrhea (US 6503951, 2000).

Derivatives of creatine and malonic, maleic, fumaric, orotic acids and taurine (CN 10/249338, 2003; US 6861554, 2005; US 6166249, 2000; CA 10/740263, 2003) are indicated for nutritional supplementation; creatine citrate (US2004077719, 2004) is recommended as a nootropic agent, as well as for use in cosmetic compositions.

Of the other creatine derivatives, the magnesium salt of creatine phosphate (CN 1709896, 2005), indicated for exposure to the heart muscle, should be noted.

Closest to the claimed substances are creatine esters, such as ethyl and benzyl (WO 02/22135, 2002) and compositions based on them, which, compared with creatine, have higher solubility in water and penetrate better through the cell membrane. The effectiveness of the creatine esters themselves has not been investigated, however, it was assumed that when they enter the blood, these derivatives under the action of enzymes (esterases) are converted to creatine. Creatine ester preparations are used as a dietary supplement orally in the form of solutions, emulsions, tablets or capsules.

The disadvantage of these compounds is the lack of stability in the body and low bioequivalence. This makes it preferable to use creatine esters in solid forms or powders and in increased daily doses.

The technical problem solved by the authors was the creation of new keratin derivatives obtained by chemical synthesis, with higher stability and a wide range of biological effects, in particular with neuroprotective effects.

At present, a large group of drugs is known that have the ability to have a specific effect on the energy metabolism of brain tissue, activate integrative functions of the brain, increase brain resistance to damaging factors (M.D. Mashkovsky. Medicines, M., Medicine; Goodman E. Gilman's The Pharmacological Basis of Therapeutics, 11 ed, McGraw-Hill, Medical Publishung Division, New York 2006; RU 1746886, 1991, WO 96/08527, 1996). These include, in particular, pyrrolidone derivatives (eg, piracetam) that activate energy metabolism; drugs that enhance cholinergic processes (e.g., amiridine, tacrine, gliatilin, etc.; GABA-energy drugs (e.g., gamma-aminobutyric acid, hopantenic acid, picamilon, phenibut), activating Krebs cycle enzymes; antioxidants and membrane protectors (e.g., mexidol , meclofenoxate, pyritino, ubiquinone); drugs of complex metabolic action (for example, vinpocetine) that optimize redox processes that help improve energy metabolism.

The disadvantages of most of these substances is a narrow spectrum of action, due to a significant number of contraindications, low neuroprotective effectiveness. At the same time, it is known that the use of effective neuroprotective agents in the clinic would increase the proportion of “small” strokes among ischemic lesions of the cerebral circulation, significantly reduce the size of the infarction zone, extend the period of the “therapeutic window”, and protect against reperfusion injury (Lancet, 2004, 363 , 349-45).

Among the drugs of this group, Actovegin, which is the closest analogue in action to the claimed drugs, received the greatest use. Actovegin contains deproteinized hemoderivative from the blood of calves, used in the form of tablets or a solution for injection (VIDAL Handbook, 2001, AstraPharmService, p. B-18).

The technical result was obtained by synthesis of creatine derivatives of the general formula NH = C (NH ₂ ) -N (CH ₃ ) -CH ₂ -CO-NH-R * X, where R is the amino acid residue or substituted amino acid residue; X is an organic or mineral acid or water.

Various aliphatic, aromatic and heteroaromatic L-amino acids or their derivatives, in particular amino acid esters, amino acid amides, peptides and the like, can be used as amino acids. As organic or mineral acids - pharmaceutically acceptable low molecular weight organic or mineral acids (molecular weight, usually less than 300), such as acetic, hydrochloric, citric, etc.

As was established during biological experiments, the synthesized creatine amides in comparison with known analogues have increased solubility and stability in aqueous solutions, which allows them to be more widely used as a source of creatine in the body.

Creatine amides were prepared by reacting free or protected guanidylating agents with sarcosine amides in polar organic solvents at a temperature not exceeding 50 ° C. The synthesis at a higher temperature, as a rule, reduces the yield of the target product and negatively affects its biological activity due to the occurrence of adverse reactions. The choice of specific synthesis conditions is determined by the characteristics of the reagents used and the resulting product.

Derivatives of amino acids can be obtained, in particular, using standard chemical reactions described in the literature (A. A. Gershkovich, V. K. Kibirev, “Synthesis of Peptides. Reagents and Methods.” Kiev. “Naukova Dumka.” 1987) or by use as a starting material creatine amides obtained by the above method.

Chemical purity analysis and semi-preparative purification of amide derivatives of creatine were performed by high performance liquid chromatography on a System Gold (Beckman) chromatograph and Phenomenex Luna C ₁₈ columns (4.6 × 150 mm, 5 μm) for analytical chromatography and Discovery C ₁₈ (10 × 250 mm, 5 microns) - for semi-preparative. Analysis conditions: UV detection at 230 nm, gradient elution in the system 0.1% trifluoroacetic acid-acetonitrile solution at a flow rate of 1 ml / min for analytical chromatography and gradient elution in the system 0.1% trifluoroacetic acid-acetonitrile solution at a flow rate of 5 ml / min for semi-preparative chromatography. Mass spectra were recorded on an MX-5303 time-of-flight mass reflectron with an Electrospray-type ion source (FINEPHF RAS).

The most promising area for the use of creatine amides is their use as substances with a neuroprotective effect. As the experiments showed, when they are introduced into the body at a dose of 20 mg / kg or more, an improvement in cognitive functions is observed, which makes creatine amides promising for the prevention and treatment of ischemic brain damage.

Creatine amides can be introduced into the body either independently or as part of compositions containing a mixture of the active principle with excipients. As excipients, substances permitted by the Pharmacopoeia are used that improve the conditions for the preparation, storage or use of a medicinal product, such as solvents, fillers, binders, disintegrants, lubricating lubricants, film formers, pigments, plasticizers, prolonging agents, flavorings, flavoring agents, stabilizers, preservatives, etc.

So, such excipients can be used such solvents as water, solutions of salts of potassium, magnesium, zinc, manganese, saline, syrup masses; fillers such as crospovidone, sugars and their derivatives, polysaccharides and their derivatives, in particular lactose, sucrose, microcrystalline cellulose, starch, glucose, mannitol, cyclodextrins, alginates, dextrin, salts of organic and mineral acids; binders such as water, ethyl alcohol, sugar syrup, starch paste, solutions of cellulose derivatives, povidones, gelatin, alginates, etc .; disintegrants such as starches, crospovidones, polysorbates, sodium lauryl sulfate, aerosil, etc .; slip lubricants such as starches, talc, polyethylene glycol, aerosil, calcium and magnesium stearates, stearic acid, sodium stearyl fumarate, and the like; film former such as alkyl celluloses and their derivatives, plasticizers such as polysorbates, glycerin, polyethylene glycol, propylene glycol, dibutyl phthalate, glycerol triacetate and the like.

The essence and advantages of the claimed group of inventions are illustrated by the following examples.

Example 1. The synthesis of creatine amide and substituted γ-aminobutyric acid - creatinyl-γ-aminobutyric acid ethyl acetate.

To a solution of 0.66 g (2.08 mmol) of sarcosyl-γ-aminobutyric acid ethyl ester trifluoroacetate in 1.5 ml of dimethylformamide was added 0.94 ml (4.16 mmol) of diisopropylethylamine and 0.9 g (2.08 mmol) of benzotriazole-1-carboxamidine tosylate. The reaction mixture was stirred for 60 hours at room temperature, diluted with n-butyl alcohol and water, and the solvent was evaporated. The residue was recrystallized from isopropyl alcohol and purified by ion exchange chromatography on a Sephadex SE C-25 column in 0.002 M pyridine acetate buffer. Fractions containing the final product were combined and the solvent was evaporated.

Yield C ₁₀ H ₂₀ N ₄ O ₃ * CH ₃ COOH 0.3 g (59%). Mass spectrum, found: m / z: 245.17.

Calculated: M 244.15.

Example 2. Synthesis of creatine amide and substituted phenylalanine - creatinyl-L-phenylalaninamide hydrate.

To a solution of 4 g (11.45 mmol) of sarkosyl-L-phenylalanine amide trifluoroacetate in 30 ml of dimethylformamide was added 3.2 ml (22.9 mmol) of triethylamine and 5.3 g (11.45 mmol) of N, N'-dibenzyloxycarbonyl-1-H-benzotriazole-1-carboxamidine . The reaction mixture was stirred for 20 hours at room temperature and diluted with 200 ml of ethyl acetate. The organic phase was washed with a 5% solution of NaHCO ₃ , water, 1 N. HCl, water and kept for 12 hours at + 4 ° C. The precipitate containing dibenzyloxycarbonyl-creatinyl-L-phenylalanine amide was filtered off, washed with cold ethyl acetate and dried in vacuo. Yield 3.8 g (62%).

To remove the protective group, 3.8 g (6.96 mmol) of dibenzyloxycarbonyl-creatinyl-phenylalanine amide was dissolved in 150 ml of dioxane-water mixture (9: 1) and hydrogenated over Pd in black for 5 hours (control by thin layer chromatography). The catalyst was filtered off, the solvent was evaporated, and the residue containing creatinyl-L-phenylalanine amide hydrate was crystallized from isopropyl alcohol. Yield C ₁₃ H ₁₉ N ₅ O ₂ * H ₂ O 1.2 g (43%). Mass spectrum, found: m / z: 278.15. Calculated: M 277.15.

Example 3. Synthesis of creatine amide and substituted phenylalanine - creatinyl-L-phenylalaninamide acetate.

The creatinyl-L-phenylalanine amide obtained under the conditions of Example 2 was dissolved in water and applied to a 25 × 100 mm column packed with Lichroprep RP-18 silica gel (43-60 μm, Merck), after which it was eluted with 0.2% acetic acid and isolated from solution.

Yield C ₁₃ H ₁₉ N ₅ O ₂ * CH ₃ COOH 0.72 g (60%). Mass spectrum, found: m / z: 278.15. Calculated: M 277.15.

Example 4. The synthesis of creatine amide and substituted glycine - creatinyl-glycine benzyl ester hydrochloride.

To a solution of 2.5 g (7.07 mmol) of sarcosyl-glycine benzyl ester trifluoroacetate in 20 ml of dimethylformamide was added 1.97 ml (14.14 mmol) of triethylamine and 2.8 g (7.07 mmol) of N, N'-di-tert-butyloxycarbonyl-1-H-benzotriazole- 1-carboxamidine and stirred for 24 hours at 20 ° C. The reaction mixture was diluted with 200 ml of ethyl acetate, the organic phase was washed with 5% solution of NaHCO ₃ , 1N H ₂ SO ₄ , water and dried over Na ₂ SO ₄ . The solvent was evaporated on a rotary evaporator and the residue was crystallized from diethyl ether - petroleum ether. Yield of di-tert-butyloxycarbonyl-creatinyl-glycine benzyl ester 2.3 g (67%). R _f 0.68 (B).

To remove the protective groups through a solution containing 2.3 g (4.78 mmol) of di-tert-butyloxycarbonyl-creatinyl-glycine benzyl ester in 70 ml of dry diethyl ether, a stream of dry HCl was passed under vigorous stirring for 45 minutes. The precipitate was filtered off, washed with dry diethyl ether and dried in a desiccator over NaOH.

Yield C ₁₃ H ₁₈ N ₄ O ₃ 0.3 g (20%). Mass spectrum, found: m / z: 279.14 Calculated: M 278.14.

Example 5. The synthesis of creatine amide and substituted tyrosine - cretinyl-tyrosinamide succinate.

To a solution of tert-butyloxycarbonylsarcosine (4.49 g, 23.74 mmol) and hydroxybenzotriazole (3.21 g, 23.74 mmol) in 30 ml of dimethylformamide, a solution of dicyclohexylcarbodiimide (4.9 g, 23.74 mmol) in 10 ml was added while cooling in an ice bath. After 10 minutes, tyrosine methyl ester hydrochloride (5 g, 21.58 mmol) and triethylamine (3.02 ml, 21.58 mmol) were added to the reaction mixture, after which they were stirred for 1 h at 0 ° С and 12 h at 20 ° С. The reaction mixture was diluted with 300 ml of ethyl acetate, washed with 5% NaHCO ₃ , 1N H ₂ SO ₄ , water and dried over Na ₂ SO ₄ . The solvent was evaporated on a rotary evaporator. R _{f of the} final product 0.75 in the system CHCl ₃ : EtOAc: MeOH 20: 10: 3. The yield of tert-butyloxycarbonylsarcosyl tyrosine methyl ester was 6.2 g (78.5%).

6.2 g (16.92 mmol) of tert-butyloxycarbonylsarcosyl tyrosine methyl ester were dissolved in 200 ml of a 6 M solution of ammonia in methanol cooled to 0 ° C. The flask was tightly closed and the solution was kept for 48 hours at room temperature. The progress of the reaction was monitored by thin layer chromatography in the CHCl ₃ : EtOAc: MeOH 20: 10: 3 system (product R _f 0.32). The solvent was evaporated, the residue was triturated with diethyl ether until a dry amorphous precipitate formed and dried in vacuum. The yield of tert-butyloxycarbonylsarcosyl tyrosinamide was 5.4 g (90%).

5.4 g of tert-butyloxycarbonylsarcosyl tyrosinamide was dissolved in 20 ml of trifluoroacetic acid, kept for 15 minutes at room temperature, the solvent was evaporated on a rotary evaporator at 25 ° С. The residue was crystallized from diethyl ether and dried in vacuo. The purity of the obtained product was checked by RP-HPLC on a Luna C-18 column 4.6 × 150 mm, 5 μm (Phenomenex) using a linear gradient of acetonitrile in water containing 0.1% phosphoric acid (0-20% acetonitrile in 20 min), flow rate 1 ml / min. The yield of sarcosyl tyrosinamide trifluoroacetate was 5.4 g (96%).

To a solution of sarcosyl tyrosinamide trifluoroacetate (5.4 g, 14.8 mmol) and tosylate benzotriazole-1-carboxamide (4.92 g, 14.8 mmol) in 7 ml of dimethylformamide were added with stirring N, N'-diisopropylethylamine (5.06 ml, 29.6 mmol). According to HPLC, after 72 hours, the completion of the reaction was about 90%. The solvent was evaporated with a mixture of water: n-butanol (2: 3), the residue was dissolved in 20 ml of water, applied to a 25 × 150 mm column with Sephadex SE C-25 (Pharmacia Fine Chemicals), equilibrated in 0.002 M pyridine-succinate buffer. Flow rate 2 ml / min. Further separation was carried out in a pyridine-succinate buffer gradient in a concentration range from 0.002 to 0.5 M. Fractions containing cretinyl tyrosinamide succinate were analyzed by RP HPLC, combined and evaporated. Final purification was carried out by crystallization from isopropyl alcohol. The purity of the obtained product was checked using RP HPLC on a Luna C-18, 4.6 × 150 mm, 5 µm column (Phenomenex) using a linear gradient of acetonitrile in water containing 0.1% phosphoric acid (0-20% acetonitrile in 20 min), speed flow 1 ml / min.

The yield of cretinyl tyrosinamide succinate 2.7 g (51%). Mass spectrum, found: m / z: 292.27. Calculated: M 292.29.

Example 6. The synthesis of creatine amide and substituted glycine - creatinyl-glycineethylamide acetate.

To a solution of tert-butyloxycarbonylsarcosyl-glycine methyl ester (15 g, 54.88 mmol) in 20 ml of dry ethanol cooled to 0 ° C, 10 ml of ethylamine was added, the flask was tightly closed and left at room temperature. According to TLC, the reaction takes place completely in 48 hours. The solvent was evaporated on a rotary evaporator. R _{f of the} product 0.45 in the CHCl ₃ : EtOAc: MeOH system (20: 10: 3).

The yield of tert-butyloxycarbonylsarcosyl-glycineethylamide was 14.7 g (98%).

14.7 g (53.85 mmol) of tert-butyloxycarbonylsarcosyl-glycineethylamide were dissolved in 40 ml of trifluoroacetic acid, kept for 15 minutes at room temperature, and the solvent was evaporated on a rotary evaporator at 25 ° С. The residue was crystallized from 150 ml of diethyl ether and dried in vacuo. The purity of the obtained product was monitored by RP HPLC on a Zorbax ODS column 250 × 4.6 mm, 5 μm (DuPont) in the mobile phase containing 0.1% TFA (0.5–20% acetonitrile in 20 min). The yield of sarcosyl glycineethyl amide trifluoroacetate was 13.9 g (92%).

To a solution of sarcosyl glycineethylamide trifluoroacetate (5 g, 17.3 mmol) and tosylate benzotriazole-1-carboxamide (5.78 g, 17.3 mmol) in 10 ml of dimethylformamide, N, N-diisopropylethylamine (6 ml, 34.6 mmol) was added with stirring. According to RP HPLC on a Zorbax ODS column 250 × 4.6 mm, 5 μm (acetonitrile gradient 0-20% in 20 minutes), after 48 hours the reaction is 90%. The solvent was evaporated with a mixture of water - n-butanol (2: 3), the residue was dissolved in 40 ml of 20% isopropyl alcohol in water and applied to a 25 × 150 mm column with Sephadex SE C-25 (Pharmacia Fine Chemicals), equilibrated in 0.002 M pyridine acetate buffer containing 20% isopropyl alcohol. Next, separation was carried out in a gradient of buffer concentration from 0.002 to 0.25 M. Fractions containing creatinyl-glycineethylamide acetate were analyzed by RP HPLC on a Zorbax ODS column of 250 × 4.6 mm, 5 μm in the mobile phase containing 0.1% TFA (0-20% acetonitrile for 20 min), combined and evaporated. The final purification of the product was carried out by crystallization from 10 ml of isopropyl alcohol at - 5 ° C. The purity of the product was monitored by RP HPLC on a Zorbax ODS column of 250 × 4.6 mm, 5 μm in the mobile phase containing 0.1% TFA (0.5–20% acetonitrile in 20 min). The yield of creatinyl-glycineethylamide acetate 2.7 g (56%). Mass spectrum, found: m / z: 214.27. Calculated: M 214.25.

Example 7. Synthesis of creatine amide and substituted phenylalanine - creatinyl-phenylalanyl-arginyl-glycine ethyl acetate.

To a solution of dicyclohexylcarbodiimide-tert-butyloxycarbonyl-ornithine (7.9 g, 21.52 mmol) and hydroxybenzotriazole (2.91 g, 21.52 mmol) in 20 ml of dimethylformamide, a solution of N, N'-dicyclohexyl 4.4 mbodiimide, 5 (21 m2) was added while stirring in an ice bath. ) in 10 ml of dimethylformamide. After 10 minutes, glycine ethyl ester hydrochloride (3 g, 21.52 mmol) and triethylamine (3 ml, 21.52 mmol) were added to the reaction mixture, stirred for 1 hour at 0 ° С and 20 hours at 20 ° С. The reaction mixture was diluted with 300 ml of ethyl acetate, washed with 5% NaHCO ₃ solution, 1N H ₂ SO ₄ , water and dried over Na ₂ SO ₄ . The solvent was evaporated on a rotary evaporator, the residue was a crystalline substance. Mp 113-115 ° C; R _f 0.89 in the system CHCl ₃ : EtOAc: MeOH 20: 10: 3. The yield of dicyclohexylcarbodiimide-tert-butyloxycarbonyl-ornityl-glycine ethyl ester was 8.6 g (88%).

To a solution of 8.6 g of dicyclohexylcarbodiimide-tert-butyloxycarbonyl-ornityl-glycine ethyl ester in 150 ml of methanol distilled over Mg was added palladium black and hydrogenated for 3 hours. The completeness of the reaction was monitored by TLC in the CHCl ₃ : EtOAc: MeOH 20: 10: 3 system. The solvent was evaporated on a rotary evaporator. The purity of the obtained product was checked by TLC in the EtOAc: n-BuOH: AcOH: H ₂ O 2: 1: 1: 1 system (R _f 0.43).

Yield of tert-butyloxycarbonyl-ornithyl-glycine ethyl ester 6 g (100%).

To a solution of dicyclohexylcarbodiimide-phenylalanine (6.24 g, 20.86 mmol) and hydroxybenzotriazole (2.82 g, 20.86 mmol) in 20 ml of dimethylformamide was added a solution of dicyclohexylcarbodiimide (4.3 g, 20.86 mmol) in 10 ml while cooling in an ice bath. After 10 minutes, tert-butyloxycarbonyl-ornithyl-glycine ethyl ether (6 g, 18.92 mmol) was added to the reaction mass, stirred for 1 hour at 0 ° С and 20 hours at 20 ° С. The reaction mixture was diluted with 300 ml of ethyl acetate, washed with 5% NaHCO ₃ solution, 1N H ₂ SO ₄ , water and dried over Na ₂ SO ₄ . The solvent was evaporated on a rotary evaporator, the product crystallized upon evaporation. Mp 179-182 ° C; R _f 0.52 in the CHCl ₃ : EtOAc: MeOH 20: 10: 3 system.

Yield of dicyclohexylcarbodiimide-phenylalanyl-ornithyl (tert-butyloxycarbonyl) -

ethyl glycine 8.7 g (77%).

To a solution of 8.7 g of dicyclohexylcarbodiimide-phenylalanyl-ornithyl (tert-butyloxycarbonyl) -glycine ethyl ester in 150 ml of methanol distilled over Mg, palladium black was added and hydrogenated for 3 hours. The completeness of the reaction was monitored by TLC in a CHCl ₃ : EtOAc: MeOH 20: 10: 3 system. The solvent was evaporated on a rotary evaporator. The purity of the obtained product was checked by TLC in the EtOAc: n-BuOH: AcOH: H ₂ O 2: 1: 1: 1 system (R _f 0.27).

The yield of phenylalanyl-ornithyl (tert-butyloxycarbonyl) -glycine ethyl ester 6.58 g (100%).

To a solution of tert-butyloxycarbonylsarcosine (3.78 g, 20 mmol) and hydroxybenzotriazole (2.7 g, 20.00 mmol) in 20 ml of dimethylformamide was added a solution of dicyclohexylcarbodiimide (4.12 g, 20.00 mmol) in 10 ml of dimethylcarbimide while cooling in an ice bath. After 10 minutes, phenylalanyl-ornithyl (tert-butyloxycarbonyl) -glycine ethyl ester (6.58 g, 14.2 mmol) was added to the reaction mixture and stirred for 1 hour at 0 ° С and 20 hours at 20 ° С. The reaction mixture was diluted with 300 ml of ethyl acetate, washed with 5% solution of NaHCO ₃ , 1N H ₂ SO ₄ , water. The solution was cooled and kept for 3 hours at 4 ° C; the product precipitated. The precipitate was filtered off, washed with ether and dried in vacuo. Mp 189-192 ° C; R _f 0.40 in the system CHCl ₃ : EtOAc: MeOH 20: 10: 3.

The yield of tert-butyloxycarbonylsarcosyl-phenylalanyl-ornithyl (tert-butyloxycarbonyl) -

ethyl glycine 6.5 g (72%).

6.5 g of tert-butyloxycarbonylsarcosyl-phenylalanyl-ornithyl (tert-butyloxycarbonyl) -

ethyl glycine was dissolved in 40 ml of trifluoroacetic acid, kept for 15 minutes at room temperature, the solvent was evaporated on a rotary evaporator at 25 ° C. The residue was crystallized from 250 ml of diethyl ether and dried in vacuo. The purity of the obtained product was checked by RP-HPLC on a Phenomenex Luna C-18, 5 µ, 4.6 × 150 mm column using a linear gradient of acetonitrile in water containing 0.1% trifluoroacetic acid (7-27% acetonitrile in 20 min), flow rate 1 ml / min

The yield of sarcosyl-phenylalanyl-ornithyl-glycine ethyl ester trifluoroacetate 6.5 g (96%).

A solution of sarcosyl-phenylalanyl-ornityl-glycine ethyl ester trifluoroacetate (2 g, 3.02 mmol) in 5 ml of water was applied to a 15 × 100 mm column with Amberlite IRA-67 ("Sigma") in the OH ^- form, and the column was washed with water. Fractions with a pH greater than 7 were combined, evaporated and dried by distillation three times with isopropyl alcohol. The resulting sarcosyl-phenylalanyl-ornityl-glycine ethyl ester was dissolved in 5 ml of dimethylformamide, 0.5 g of LiCl (Merck) was added to improve solubility, after which N, N'-diisopropylethylamine (1.05 ml, 6.04 mmol) and benzotriazole-1-carboxamide tosylate were added. (2.01 g, 6.04 mmol), 5 ml of dimethylformamide was added and stirred for 72 hours. According to RP HPLC on a Phenomenex Luna C-18 column, 5 µ, 4.6 × 150 mm, a linear gradient of acetonitrile in water containing 0.1% trifluoroacetic acid (7-27% acetonitrile in 20 min, flow rate 1 ml / min), reaction passes more than 90% after 40 hours. The solvent was evaporated with a mixture of water - n-butanol (2: 3), the residue was dissolved in 20 ml of water and applied to a 25 × 150 mm column with Sephadex SE C-25 (Pharmacia Fine Chemicals), balanced in 0.002 M pyridine-acetate buffer. The column was washed with 400 ml of 0.002 M buffer (flow rate of 2 ml / min), separation was carried out in a gradient of 0.002-0.5 M of pyridine acetate buffer. Fractions containing creatinyl-phenylalanyl-arginyl-glycine ethyl ether acetate were combined and evaporated. The final purification of the product was carried out by crystallization from 20 ml of isopropyl alcohol at room temperature. The purity of the obtained product was checked by RP HPLC on a Luna C-18, 5 μm, 4.6 × 150 mm column (Phenomenex) using a linear gradient of acetonitrile in water containing 0.1% phosphoric acid (8-28% acetonitrile in 20 min), speed flow 1 ml / min.

Yield of creatinyl-phenylalanyl-arginyl-glycine ethyl acetate 0.72 g (37%). Mass spectrum, found: m / z: 520.60. Calculated: M 520.61.

Example 8. The synthesis of creatine amide and unsubstituted phenylalanine - creatinyl-phenylalanine hydrochloride.

To a solution of tert-butyloxycarbonylsarcosine (4 g, 21.14 mmol) and hydroxybenzotriazole (2.86 g, 21.14 mmol) in 30 ml of dimethylformamide was added a solution of dicyclohexylcarbodiimide (4.36 g, 21.14 mmol) in 10 ml of dimethylformamide while cooling. After 10 min, p-toluenesulfonic acid benzyl ester (8.13 g, 19.03 mmol) and triethylamine (2.7 ml, 19.03 mmol) were added to the reaction mass, stirred for 1 h at 0 ° С and 20 h at 20 ° С. N, N'-Dicyclohexylurea was filtered off, the reaction mixture was diluted with 200 ml of ethyl acetate, washed with 5% NaHCO ₃ solution, 1N H ₂ SO ₄ , water and dried over Na ₂ SO ₄ . The solvent was evaporated on a rotary evaporator. R _{f of the} basic substance is 0.85 in the CHCl ₃ : EtOAc: MeOH system (20: 10: 3).

The yield of tert-butyloxycarbonylsarcosyl-phenylalanine benzyl ether 7.3 g (90%).

7.3 g (17.15 mmol) of tert-butyloxycarbonylsarcosyl-phenylalanine benzyl ether was dissolved in 20 ml of 65% trifluoroacetic acid in CH ₂ Cl ₂ , held for 30 minutes at room temperature, and the solvent was evaporated on a rotary evaporator at 25 ° C. The residue was crystallized from 200 ml of diethyl ether, dried in vacuum. The purity of the obtained product was checked using RP HPLC on a Phenomenex Luna C-18 column, 5 μm, 4.6 × 150 mm (20-40% acetonitrile in 20 minutes).

The yield of sarcosyl-phenylalanine benzyl ester trifluoroacetate 6 g (80%).

To a solution of sarcosyl-phenylalanine benzyl ester trifluoroacetate (6 g, 13.62 mmol) and benzotriazole-1-carboxamide tosylate (4.54 g, 13.62 mmol) in 7 ml of dimethylformamide was added with stirring N, N'-diisopropylethylamine (4.74 ml, 27.24 mmol). According to RP HPLC on a Phenomenex Luna C-18 column, 5 μm, 4.6 × 150 mm (20-40% acetonitrile in 20 minutes), after 72 hours the reaction was almost complete. The solvent was evaporated with a mixture of water - n-butanol (2: 3), the residue was dissolved in 80 ml of 30% aqueous isopropyl alcohol and applied to a 25 × 150 mm column with Sephadex SE C-25 (Pharmacia Fine Chemicals), equilibrated in 0.002 M pyridine -acetate buffer containing 20% isopropyl alcohol. The column was washed with 400 ml of start buffer (flow rate of 2 ml / min), separation was carried out in a gradient of 0.002-0.5 M pyridine acetate buffer containing 20% isopropyl alcohol.

Fractions containing AcOH * Cr-Phe-OBzl were analyzed by RP HPLC on a Phenomenex Luna C-18 column, 5 μm, 4.6 × 150 mm (20-40% acetonitrile in 20 min), combined and evaporated.

The yield of creatinyl-phenylamine benzyl ether acetate 2.54 g (41%).

2.54 g (5.93 mmol) of creatinyl-phenylamine benzyl ester acetate was dissolved in 80 ml of methanol and hydrogenated over Pd black. As the reaction progressed, the product precipitated. After 4 hours, according to TLC in the ACN: H ₂ O: AcOH (7: 1: 1) system, the hydrogenation was almost complete. The precipitate formed together with the catalyst was filtered off, washed with methanol and dissolved in 200 ml of 0.01 M HCl in 40% aqueous methanol. The catalyst was filtered off, the filtrate was evaporated, the residue was crystallized from 10 ml of isopropyl alcohol. The purity of the obtained product was checked using RP HPLC on a Luna C-18 column, 5 μm, 4.6 × 150 mm, Phenomenex (5-25% acetonitrile in 20 min).

Yield 1.1 g (69%). Mass spectrum, found: m / z: 278.27. Calculated: M 278.29.

Example 9. The study of the stability of creatine amides in aqueous solution and blood plasma.

The stability of creatine derivatives in aqueous solution, as well as human and rat blood plasma, was studied by reverse phase HPLC using a Beckman System Gold chromatographic system (USA) in the following configuration: Programmable Solvent Module 126, Manual Injector Rheodyne 7725i, Programmable Detector Module 166, equipped with Beckman System Gold Chromatography Software data management and processing software. The analysis was performed on a Luna 5µ, C 18 (2) 100 Å 150 × 4.6 mm column (Phenomenex, USA) with a Guard Cartridge C 18 pre-column at a flow rate of 1 ml / min in a linear acetonitrile gradient: from 5% to 5% for 25% acetonitrile in 20 minutes (buffer A: 0.1% H ₃ PO ₄ -H ₂ O, buffer B: 0.1% H ₃ PO ₄ — acetonitrile), in the case of the substance from example 2-8, from 3% to 23% acetonitrile in 20 minutes Detection was carried out at a wavelength of 220 nm. The dosage of the samples on the column was carried out with a loop of 20 μl.

For the preparation of solutions of the studied substances on an analytical balance, an exact weight of each peptide was taken. To it was added the calculated amount of double-distilled water to obtain a concentration of 2 mg / ml. Part of the solution was diluted 10 times and the sample was immediately analyzed. Then this solution was kept at room temperature and after 3 hours the analysis was repeated. According to the results of these determinations, the peak area of both drugs after 3 hours decreases slightly (less than 3%), the appearance of new peaks was not detected.

To study the stability of creatine amides, 1 ml of water or blood plasma was added to 200 μl of a solution of the starting amide in water with a concentration of 2-3 mg / ml, shaken, and a sample of 200 μl was immediately taken and the initial concentration of creatine amide was analyzed. Next, the solution was placed in a vibration thermostat at a temperature of 37 ° C and an aliquot of 200 μl was taken from it after 0.5, 1 and 3 hours of temperature control. 20 μl of a 10% trichloroacetic acid solution was added to the selected sample and kept for 15 min at a temperature of minus 24 ° С, centrifuged at 6000 g for 5 min to precipitate plasma proteins, the supernatant was taken and analyzed. To assess the stability of the preparations, the peak areas of the corresponding compound were compared at the beginning of the experiment and at selected time intervals (tables 1-2).

Table 1
Stability of creatine analogues in human blood plasma A drug Stability in human plasma 0 h 0.5 h 0.75 h 1 hour 3 h According to example 1 one hundred% 99% - 103% 104% According to example 2 one hundred% 99% - 99% one hundred% For example 3 one hundred% one hundred% - one hundred% one hundred% For example 4 one hundred% 98% - one hundred% 99% For example 5 one hundred% 98% - 97% 93% For example 6 one hundred% 97% - 95% 95% For example 7 one hundred% 94% - 90% 85% For example 8 one hundred% 99% - 98% 97% Creatine Benzyl Ether one hundred% - 53% - 46% table 2
Stability of creatine analogues in rat plasma A drug Rat plasma stability 0 h 0.5 h 1 hour 3 h According to example 1 one hundred% 96% 98% 99% According to example 2 one hundred% one hundred% 97% 98% For example 3 one hundred% 96% 98% 99% For example 4 one hundred% 99% 99% 96% For example 5 one hundred% 98% 97% 93% For example 6 one hundred% 97% 97% 95% For example 7 one hundred% 93% 92% 85% For example 8 one hundred% 98% 98% 95%

As follows from the above data, creatine amides have high stability in the plasma of both humans and rats: their concentration for 3 hours remained almost unchanged, while the concentration of the drug analogue - creatine benzyl ester - in human blood plasma for 0.75 hours decreased by 53% in all cases.

Example 10. Nootropic effect of creatine amides.

A study of the effect of creatine amides on their ability to exhibit a neuroprotective effect on brain tissue was performed on a model of focal and global cerebral ischemia of rats.

Global cerebral ischemia was induced in male S-D rats anesthetized with Nembutal by clamping both carotid arteries for 12 min with simultaneous controlled hypotension (45 mmHg).

The intracerebroventricular (ICC) cannula was stereotactically injected into the left lateral cerebral ventricle using ketamine anesthesia and attached to an Alzet osmotic mini-pump (AP) (model 1002, the flow rate of the solution was 0.25 μl / h), which was previously filled with experimental solution and placed subcutaneously between the shoulder blades.

The studied drugs were introduced (a, b) or orally (a) in doses of 20 mg / kg weight, in the following experimental variants:

(a) - starting 5 days before ischemia and continuing 7 days after ischemia (assessment of the effectiveness of the drug for the prevention and treatment of ischemia);

(b) - starting 30 minutes after the induction of ischemia and continuing for 7 days (evaluation of the effectiveness of the drug for the treatment of ischemia);

(c) - starting 1 hour before ischemia (assessment of the effectiveness of the drug for the prevention and treatment of ischemia).

On the 7th day after ischemia, rats were sacrificed by decapitation, the brain was removed, immersed for 2-24 hours in 2% paraformaldehyde, then in 96% ethanol, encoded for a “blind” analysis, and a morphological study was performed. The degree of brain damage was assessed by the number of “compressed” neurons in different regions of the brain.

In general, a conclusion on the effectiveness of the drugs was made on the basis of a study of their effect on the behavioral reactions of animals in the Morris water maze and on the basis of the analysis of morphological changes in brain tissue with the introduction of creatine amides against the background of global and focal cerebral ischemia.

1. Data on behavioral testing in the Morris water maze (TMV) with intraperitoneal administration of the drug.

For the study, the composition of the preparation of the following composition was used (table 3).

Table 3
The composition of the compositions for biological research No. Component Name Amount, g Composition 1 one Amide creatine in example 1 20,0 Sodium chloride 9.0 Nipagin 1,0 Sodium hydroxide 0.1 M solution up to pH 7, Water up to 1 l Composition 2 2 Amide creatine according to example 2 10.0 Magnesium sulfate 10.0 Nipagin 1,0 Sodium hydroxide 0.1 M solution to pH 7.2 Water up to 1 l Composition 3 3 Amide creatine in example 1 30,0 Succinic acid 0.5 Nipagin 1,0 Potassium hydroxide 0.1 M solution to pH 7.2 Water up to 1 l Composition 4 four Amide creatine according to example 3 0.35 Microcrystalline cellulose 0,0769 Crospovidone 0,022 Calcium Phosphate Dihydrate 0.1000 Sodium Stearyl Fumarate 0.1000 Total 0.65 Composition 5 5 Amide creatine according to example 4 0.35 Crospovidone 0.017 Lactose 0.003 Stearic acid 0.002 Potassium citrate 0.002 Aerosil 0.001 Total 0.375 Composition 6 6 Amide creatine according to example 4 0.35 Mannitol 0.006 Aerosil 0.001 Total 0.357 Composition 7 7 Actovegin (analogue in action) 0.35 Microcrystalline cellulose 0,0769 Crospovidone 0,022 Calcium Phosphate Dihydrate 0,100 Sodium Stearyl Fumarate 0,100 Total 0.65 Composition 8 8 Amide creatine according to example 5 2.0 Sodium chloride 9.0 Nipagin 1,0 Sodium hydroxide 0.1 M solution to pH 7.2 Water up to 1 l Composition 9 9 Amide creatine according to example 7 2.0 Sodium chloride 9.0 Nipagin 1,0 Sodium hydroxide 0.1 M solution to pH 7.2 Water up to 1 l Composition 10 10 Amide creatine according to example 6 20,0 Sodium chloride 9.0 Nipagin 1,0 Sodium hydroxide 0.1 M solution to pH 7.2 Water up to 1 l

When analyzing the effectiveness in relation to disorders of energy tissue metabolism caused by focal cerebral ischemia, we compared the training data in the TMV of rats with intravenous injection and oral administration of drugs (n = 7), negative control with intravenous administration of physiological saline (n = 13 ), positive control (n = 5) and falsely operated animals (LO) (n = 5).

Statistical analysis (ANOVA method with repeated measurements) showed that the groups differ significantly in the course of training. Training was observed in the experimental group (drug administration), the positive control group and the falsely operated animals. FIM per se completely blocked learning. Rats treated with the drug spent statistically significantly less time searching for a platform than rats of the negative control group in the last two days of training (p <0.02 and p <0.03, respectively; Fisher's post hoc LSD test) and did not differ from the positive group control and falsely operated animals (table 4).

Table 4.
Rat behavioral testing data in the Morris water maze Training day / Platform search time, sec one 2 3 four 5 Composition 1, I.C. input, experiment model a) 52 ± 5 44 ± 3 42 ± 3 40 ± 2 35 ± 3 Composition 2, I.C. input, experiment model b) 50 ± 5 43 ± 3 43 ± 3 39 ± 2 34 ± 3 Composition 3, I.C. input, experiment model b) 47 ± 4 45 ± 3 41 ± 4 39 ± 3 35 ± 3 Composition 4, oral, experimental model a) 55 ± 3 45 ± 5 42 ± 3 41 ± 3 34 ± 4 Composition 5, oral, experiment model a) 52 ± 3 45 ± 5 40 ± 3 40 ± 2 33 ± 4 Composition 6, oral, experimental model a) 52 ± 3 46 ± 5 41 ± 3 40 ± 2 32 ± 4 Composition 7, oral, experimental model a) 52 ± 3 55 ± 5 53 ± 3 53 ± 2 50 ± 5 Focal ischemia without therapy 52 ± 4 54 ± 5 53 ± 5 53 ± 3 52 ± 4 Falsely Operated Animals 32 ± 12 29 ± 15 27 ± 12 18 ± 7 12 ± 5 Focal Ischemia + Hypothermia 62 ± 4 57 ± 5 55 ± 7 45 ± 5 35 ± 4

The introduction of compositions containing creatine amides significantly reduced the latency of movement initiation in the early stages after the induction of PIM - after 1 and 3 days. According to this effect, the group with the introduction of the studied drug approaches the group of positive control (hypotremia) and the group of animal LO.

When analyzing animal behavior data in the Open Field installation, a pronounced effect of i.v. administration of creatine amides on one of the indicators of the animal's functional state in chronic PIM was noted — latency of movement initiation, reflecting the degree of disturbance in the structure of neurons and interneuronal connections. Data on latency of movement initiation rats of the group of rats with iv injection of the composition (n = 7), negative (n = 13), positive controls (n = 5) and falsely operated animals (n = 5) were compared one day before and after 1 3 and 7 days after FIM.

Initiation of movement (warm-up) is a complex multi-stage motor reaction of an organism to internal and / or external stimuli.

In accordance with the procedure proposed by Golani et al (Golani I., Wolgin DL, Teitelbaum P., 1979), to estimate the initiation of movement, the time required for the animal to go beyond the bounds of the space — a 20 × 20 cm square located in the center of the open field installation (1 × 1 × 0.5 m).

The results are presented in table 5.

Table 5
The data of behavioral testing of animals in the "open field" (composition 2, experiment a) Impact Latency of motion initiation, s 1 day before ischemia 1 day after ischemia 3 days after ischemia 7 days after ischemia FIM 9 ± 2 49 ± 4 46 ± 4 16 ± 3 FIM + composition 2 9 ± 2 25 ± 6 30 ± 8 10 ± 2 FIM + hypothermia 8 ± 2 10 ± 8 5 ± 2 2 ± 2

The introduction of creatine amides leads to a significant decrease in the area of brain damage during experimental ischemia (table 6).

Table 6
Rat brain damage in experimental ischemia / postischemic reperfusion following administration of creatine amides Drug, number of animals (n) The ratio of the area of damage to the area of the entire frontal section of the brain (%) Control, saline, and.ts.v. input, experiment model (s), (n = 6) 17.0 ± 1.8 Composition 8, i.c.v. input, experiment model (s), (n = 5) 10.3 ± 1.0 Composition 9, I.C. input, experiment model (s), (n = 5) 11.9 ± 2.0 Composition 10, I.C. input, experiment model (s), (n = 6) 8.4 ± 0.7

The data presented indicate a neuroprotective effect of drugs containing creatine amides in conditions of cerebral ischemia. Compositions based on creatine amides have a positive effect on the restoration of cognitive functions after an experimental stroke and latency of movement initiation, which characterizes the general state of energy tissue metabolism of animal brain tissue, the functional state of neurons and associative areas of the cerebral cortex,

Claims (3)

1. Creatine amides of the general formula: NH = C (NH ₂ ) -N (CH ₃ ) -CH ₂ -CO-NH-R * X, where R is the amino acid residue of an aliphatic, aromatic or heteroaromatic amino acid or its derivative, which is pharmaceutically acceptable amino acid salts, amino acid esters, amino acid amides or peptides; X is a low molecular weight organic or mineral acid or water. 2. The method of producing creatine amides according to claim 1, which consists in the fact that the reaction of guanidylating agents with sarcosine amides is carried out in polar organic solvents at a temperature of not more than 50 ° C. 3. Creatine amides of the general formula: NH = C (NH ₂ ) -N (CH ₃ ) -CH ₂ -CO-NH-R * X, where R is the amino acid residue of an aliphatic, aromatic or heteroaromatic amino acid or its derivative, which is pharmaceutically acceptable amino acid salts, amino acid esters, amino acid amides or peptides; X - low molecular weight organic or mineral acid or water, as a means of having a neuroprotective effect.